CN110343710B - Chimeric Antigen Receptors and Methods for Treating Liver Cancer - Google Patents

Abstract

The present invention provides novel chimeric antigen receptors. The present invention provides expression vectors and host cells for expressing the chimeric antigen receptor. The present invention also provides the use of chimeric antigen receptors in treating cancer or preparing drugs for treating cancer. The chimeric antigen receptor and medicine provided by the invention can effectively treat cancers such as liver cancer.

Description

Chimeric Antigen Receptors and Methods for Treating Liver Cancer

This application claims priority to the Chinese patent application filed on April 4, 2018 with application number 201810299324.8 and the invention title "NKG2D Chimeric Antigen Receptor and Methods for Treating Cancer", the entire content of which is incorporated herein by reference. Applying.

Technical field

The present invention relates to the fields of immunology and medicine. Specifically, the present invention provides novel chimeric antigen receptors. The present invention also provides the use of the new chimeric antigen receptor in treating cancer or preparing drugs for treating cancer.

Background technique

Primary liver cancer is one of the most common forms of cancer in the world. Hepatocellular carcinoma (also called malignant hepatoma) is the most common form of primary liver cancer and forms within liver cells. Hepatocellular carcinoma occurs primarily in men and in patients suffering from cirrhosis of the liver. Many patients with hepatocellular carcinoma remain asymptomatic until the disease is in its advanced stages, resulting in ineffective treatments and poor prognosis; most patients with unresectable hepatocellular carcinoma die within one year.

NKG2D (natural-killer group 2 member D, or NKG2D receptor), killer cell lectin-like receptor subfamily K member 1, is a type II transgene expressed on all natural killer cells, natural killer T cells and γδ ⁺ T cells. Membrane Protein. In humans, NKG2D receptors mainly bind to two ligands, namely UL16-binding protein (ULBP) and MHC class I-chain associated protein A/B (MHC class I-chain -related protein, MICA/B). Expression of human NKG2D ligands is rarely detected in healthy tissues but is upregulated in tumor cells and during cellular stress and viral infection. The binding of NKG2D and its ligands serves as activating signals in immune responses against tumors and viral infections.

In recent years, unprecedented results have been obtained in autologous treatment of B-cell lymphoma and leukemia using genetically engineered chimeric antigen receptor (CAR) T cells, and this approach has begun to be applied to solid tumors. CAR-modified T cells combine the HLA-independent targeting specificity of monoclonal antibodies with the cytolytic activity, proliferation, and homing properties of activated T cells but do not respond to checkpoint inhibition. However, due to their ability to directly kill target-expressing antigens, CAR T cells are highly toxic to any antigen-positive cells or tissues, which necessitates the construction of CARs through highly tumor-specific constructs.

In 2005, the NKG2D receptor-NKG2D ligand system was used for chimeric antigen receptor (CAR) therapy for the first time (T. Zhang et al., Blood, vol. 106, no. 5, pp. 1544–1551, Sep. 2005). In natural killer cells and T cells, DNAX activating protein 10 (DAP10) is the cell surface adapter of the NKG2D receptor.

Treatment options for liver cancer are limited, especially in the case of advanced or recurrent liver cancer. Surgery and radiation therapy are options for early-stage liver cancer, but are not very effective for advanced or recurrent liver cancer. Systemic chemotherapy has not yet become particularly effective, and there are a very limited number of drugs available. The currently approved kinase inhibitor sorafenib has been shown to be effective in the treatment of liver cancer. However, it can slow or stop the progression of advanced liver cancer for just a few months compared to no treatment.

Therefore, there is a need in the art for more effective and safer methods for treating liver cancer.

Contents of the invention

The invention provides a new chimeric antigen receptor, which contains: (a) an antigen-binding domain, which includes NKG2D or an active fragment thereof; (b) a transmembrane domain and (c) an intracellular signaling domain . The present invention also provides a combination of a new chimeric antigen receptor and its accessory protein DAP10 or its active fragment. The present invention also provides nucleic acids and expression vectors encoding the chimeric antigen receptor and/or DAP10, as well as cells expressing the chimeric antigen receptor and/or DAP10. The present invention also provides the use of the chimeric antigen receptor and/or DAP10, its expression vector and cells in methods of treating cancer and in preparing drugs for treating cancer.

The invention provides a chimeric antigen receptor (CAR), which includes: (a) an antigen-binding domain, which includes NKG2D or an active fragment thereof; (b) a transmembrane domain and (c) an intracellular signaling structure area.

The invention also provides a combination of the aforementioned chimeric antigen receptor (CAR) and an auxiliary protein, wherein the CAR comprises: (a) an antigen-binding domain including NKG2D or an active fragment thereof; ( b) a transmembrane domain and (c) an intracellular signaling domain, and wherein the accessory protein is DAP10 or an active fragment thereof. In one aspect of the invention, the DAP10 has the amino acid sequence of SEQ ID NO: 4.

"NKG2D" or "NKG2D receptor", also known as "NKG2-D", "CD314", "KLRK1", "killer cell lectin-like receptor subfamily K member 1", refers to the killing ability of mammals, especially humans. A cell-activating receptor gene (its mRNA such as NCBI RefSeq NM_007360) or its gene product (such as NCBI RefSeq NP_031386) or a naturally occurring variant thereof. In human NK cells and T cells, the ligand-bound form of the NKG2D receptor is a homodimer (Li et al., Nat Immunol 2001;2:443-451). NKG2D activity, including cell activation, antibody recognition, etc., also includes binding to two ligands, which are UL16-binding protein (ULBP) and MHC type I chain-related protein A/B ( MHC classI-chain-related protein MICA/B).

DAP10 (membrane protein 10) refers to the surface protein gene or its gene product of mammals, especially humans (as shown in GenBank: AAG29425.1). The activity of DAP10 involves forming a complex with NKG2D (Wu, J. et al., Science 285 (5428), 730-732, 1999).

In one aspect of the invention, the antigen-binding domain of the chimeric antigen receptor (CAR) comprises an active fragment of NKG2D. The active fragment is, for example, the a.a.82-216 fragment of NKG2D, that is, the 82-216th amino acid with the amino acid sequence of SEQ ID NO: 2: LFNQEVQIPLTESYCGPCPKNWICYKNNCYQFFDESKNWYESQASCMSQNASLLKVYSKEDQDLLKLVKSYHWMGLVHIPTNGSWQWEDGSILSPNLLTIIEMQKGDCALYASSFKGYIENCSTPNTYICMQR TV.

In one aspect of the invention, the antigen-binding domain may comprise a leading peptide. The leader sequence can assist in the expression of proteins on the cell membrane or in and out of the membrane. Leader sequences known in the art can be used in the CARs of the invention. In the present invention, the leader sequence may be located upstream of the NKG2D or active fragment thereof. In one embodiment of the present invention, the leader sequence is a leader sequence of CD33, which has the amino acid sequence of SEQ ID NO: 18. The leader sequence can promote the expression of CAR on the cell surface, but the presence of the leader sequence in the expressed CAR is not required for the CAR to function. In embodiments of the invention, the leader sequence can be cleaved from the CAR after the CAR is expressed on the cell surface. Therefore, in embodiments of the invention, a CAR may be devoid of a leader sequence.

In one aspect of the invention, the CAR includes a transmembrane domain. Transmembrane domains known in the art can be used in the present invention. Transmembrane domains include T cell receptor α, β, or ζ, CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154 ,KIRDS2,OX40,CD2,CD27,LFA-1(CD11a,CD18),ICOS(CD278),4-1BB(CD137),GITR,CD40,BAFFR,HVEM(LIGHTR),SLAMF7,NKp80(KLRF1),CD160, CD19,IL2Rβ,IL2Rγ,IL7Rα,ITGA1,VLA1,CD49a,ITGA4,IA4,CD49D,ITGA6,VLA-6,CD49f,ITGAD,CD11d,ITGAE,CD103,ITGAL,CD11a,LFA-1,ITGAM,CD11b,ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, TNFR2, DNAM1(CD226), SLAMF4(CD244,2B4), CD84, CD96(Tactile), CEACAM1, CRTAM, Ly9(CD229), CD160(BY55) , PSGL1, CD100 (SEMA4D), SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, PAG/Cbp, NKp44, NKp30, NKp46, etc. transmembrane domain.

In one aspect of the invention, the transmembrane domain of the CAR of the invention comprises i) the transmembrane domain of CD8 and/or ii) CD28. In yet another aspect of the present invention, the transmembrane domain of the CAR of the present invention is the transmembrane domain of CD28, for example, it may have the amino acid sequence of SEQ ID NO: 8.

In one aspect of the invention, the CAR includes an intracellular signaling domain. Examples of intracellular signaling domains that may be used in the present invention include those from CD2, CD4, CD5, CD8α, CD8β, CD28, CD134, CD137, ICOS, and CD154. Specific examples thereof include peptides having the following sequences: amino acid numbers 236-351 of CD2 (NCBI RefSeq: NP_001758.2), amino acid numbers 421-458 of CD4 (NCBI RefSeq: NP_000607.1), CD5 (NCBI RefSeq: NP_055022.2 ), the amino acid number of CD8α (NCBI RefSeq: NP_001759.3) is 207-235, the amino acid number of CD8β (GenBank: AAA35664.1) is 196-210, the amino acid number of CD28 (NCBI RefSeq: NP_006130.1) Numbers 181-220 (SEQ ID NO::25), amino acid numbers 214-255 of CD137 (4-1BB, NCBI RefSeq: NP_001552.2), amino acid numbers 241-277 of CD134 (OX40, NCBI RefSeq: NP_003318.1) and The amino acid numbers of ICOS (NCBI RefSeq: NP_036224.1) are 166-199, etc., and their variants have the same functions as these peptides.

Preferably, the intracellular T cell signaling domain of the CAR of the present invention includes any one or more of the following: i) CD28, ii) 4-1BB, and/or iii) the intracellular signaling domain of CD3ζ. In a preferred embodiment, the intracellular T cell signaling domain of the CAR of the present invention is the intracellular signaling domain of CD28, 4-1BB and CD3ζ. More preferably, the intracellular T cell signaling domain of the CAR of the present invention is CD28, 4-1BB and CD3ζ in order from the amino terminus to the carboxyl terminus. Among them, CD28 is an important T cell marker in T cell costimulation, and the sequence of its intracellular signaling domain may include, for example, the amino acid sequence shown in SEQ ID NO: 10. 4-1BB, also known as CD137, delivers potent costimulatory signals to T cells, thereby promoting T lymphocyte differentiation and enhancing their long-term survival. CD3ζ associates with TCRs to generate signals and contains an immunoreceptor tyrosine-based activation motif (ITAM). In one aspect of the present invention, the intracellular T cell signaling domain of the CAR includes the intracellular signaling domains of CD28, 4-1BB and CD3ζ, respectively having SEQ ID NO: 10, SEQ ID NO: 12, SEQ Amino acid sequence of ID NO:14. In yet another aspect of the present invention, the intracellular T cell signaling domain of the CAR comprises the intracellular signaling domain of CD28 having the amino acid sequence of, for example, SEQ ID NO: 10. In yet another aspect of the present invention, the intracellular signaling domain of 4-1BB comprised by the intracellular T cell signaling domain of the CAR has, for example, the amino acid sequence of SEQ ID NO: 12. In yet another aspect of the present invention, the intracellular T cell signaling domain of the CAR comprises the intracellular signaling domain of CD3ζ having the amino acid sequence of, for example, SEQ ID NO: 14.

In CARs containing multiple intracellular signaling domains, oligopeptide linkers or polypeptide linkers can be inserted between the intracellular domains to connect the domains. Preferably, linkers with a length of 2-10 amino acids may be used. In particular, linkers with glycine-serine contiguous sequences can be used.

In one embodiment of the invention, the CAR comprises (a) an antigen-binding domain, which is the a.a.82-216 fragment of NKG2D; (b) a transmembrane domain of CD28 and (c) an amino-terminal The order is the intracellular signaling domains of CD28, 4-1BB and CD3ζ.

In one aspect of the present invention, in the CAR, there is a hinge region between (a) the antigen domain and (b) the transmembrane domain. Hinge regions known in the art may be used in the present invention, including IgG1H, IgG2H, IgG3H, and IgG4H. In yet another aspect of the present invention, the hinge region between (a) the antigenic domain and (b) the transmembrane domain comprises IgG1H or a fragment or variant thereof, the amino acid sequence of which is, for example, SEQ ID NO: 6.

Included within the scope of the invention are functional variants of the proteins of the invention described herein, such as CAR or functional fragments thereof (including antigen-binding domain, transmembrane domain, intracellular signaling domain, hinge region, leader sequence, etc.). The term "functional variant" as used herein refers to a CAR, polypeptide or protein that has substantial or significant sequence identity or similarity to a parent protein, such as a CAR, that retains the biological activity of the CAR variant. Functional variants encompass, for example, those variants of a CAR described herein (parental CAR) that retain the ability to recognize a target to a similar extent to the parental CAR, to the same extent as the parental CAR, or to a higher extent than the parental CAR. cell. With respect to the parent CAR, the amino acid sequence of the functional variant may, for example, have at least about 30%, about 50%, about 75%, about 80%, about 90%, about 98%, about 99%, or Higher identity.

Functional variants may, for example, comprise the amino acid sequence of a parent CAR with at least one conservative amino acid substitution. Alternatively or additionally, a functional variant may comprise the amino acid sequence of the parent CAR with at least one non-conservative amino acid substitution. In this case, non-conservative amino acid substitutions that do not interfere with or inhibit the biological activity of the functional variant are preferred. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, making the biological activity of the functional variant increased compared with the parental CAR.

Embodiments of the invention also provide isolated nucleic acids comprising a nucleotide sequence encoding any of the CARs described herein. Nucleic acids of the invention may comprise nucleotide sequences encoding any leader sequence, antigen binding domain, transmembrane domain, and/or intracellular T cell signaling domain described herein.

In one aspect of the invention, there is provided an isolated nucleic acid comprising a nucleotide sequence encoding a chimeric antigen receptor (CAR) as described above, said CAR comprising: (a) an antigen-binding domain, comprising NKG2D or active fragment thereof; (b) transmembrane domain and (c) intracellular signaling domain,

In yet another aspect of the invention, the nucleotide sequence encoding the antigen-binding domain of the CAR includes an active fragment encoding NKG2D, preferably a nucleotide sequence encoding the a.a.82-216 fragment of NKG2D, for example, it has The nucleotide sequence shown in SEQ ID NO:1.

In yet another aspect of the invention, the nucleotide sequence encoding the transmembrane domain includes a nucleotide sequence encoding the transmembrane domain of CD8 and/or CD28, preferably the transmembrane domain of CD28, for example is the nucleotide sequence of SEQ ID NO:7.

In yet another aspect of the invention, the nucleotide sequence encoding the intracellular signaling domain includes nucleotides encoding one or more of the intracellular signaling domains of CD28, 4-1BB and CD3ζ. The sequence preferably includes a nucleotide sequence encoding the intracellular signaling domain of CD28, 4-1BB and CD3ζ, and more preferably includes a nucleic acid sequence encoding a protein that is CD28, 4-1BB and CD3ζ in order from the amino terminus to the carboxyl terminus. ;

In yet another aspect of the present invention, the nucleic acid encoding the intracellular signaling domain of CD28 has a nucleotide sequence such as SEQ ID NO: 9;

In yet another aspect of the present invention, the intracellular signaling domain encoding 4-1BB has a nucleotide sequence such as SEQ ID NO: 11;

In yet another aspect of the present invention, the intracellular signaling domain encoding CD3ζ has a nucleotide sequence such as SEQ ID NO: 13;

In yet another aspect of the present invention, the nucleotide sequence encoding the intracellular signaling domain of the CAR has a nucleotide sequence such as SEQ ID NO: 20, which includes the nucleotide sequence encoding CD28, 4-1BB and CD3ζ. Nucleotide sequence of the internal signaling domain.

In yet another aspect of the present invention, the nucleic acid provided by the present invention also includes a nucleotide sequence encoding a hinge region between (a) the antigenic domain and (b) the transmembrane domain, preferably a nucleic acid sequence encoding IgG1. A nucleotide sequence having, for example, the nucleotide sequence of SEQ ID NO:5.

In yet another aspect of the present invention, the nucleic acid provided by the present invention also includes a nucleotide sequence encoding a leader sequence located upstream of the NKG2D or its active fragment, preferably a nucleotide sequence encoding a leader sequence of CD33, for example It is the nucleotide sequence shown in SEQ ID NO: 17.

As mentioned above, the present invention also provides a combination of the previously defined chimeric antigen receptor (CAR) and an auxiliary protein, the auxiliary protein being DAP10 or an active fragment thereof.

In yet another aspect of the present invention, the nucleic acid provided by the present invention also includes a nucleotide sequence encoding the chimeric antigen receptor (CAR) and the accessory protein, and the CAR includes: (a) an antigen-binding domain, It includes NKG2D or an active fragment thereof; (b) a transmembrane domain and (c) an intracellular signaling domain, and the accessory protein is DAP10 or an active fragment thereof. Wherein, the nucleotide sequence encoding the antigen-binding domain of the CAR includes the nucleotide sequence encoding the active fragment of NKG2D, for example, the nucleotide sequence of the a.a.82-216 fragment of NKG2D, for example, the nucleoside shown in SEQ ID NO: 1 acid sequence. And, wherein the nucleic acid encoding said DAP10 has the nucleotide sequence of SEQ ID NO: 3.

As used herein, "nucleic acid" includes "polynucleotide," "oligonucleotide," and "nucleic acid molecule" and generally means a polymer of DNA or RNA, which may be single- or double-stranded, synthesized from natural sources or obtained (e.g., isolated and/or purified), may contain natural, non-natural, or altered nucleotides, and may contain natural, non-natural, or altered inter-nucleotide linkages, such as phosphoramidate linkages or phosphorothioate bonds replace the phosphodiesters present between the nucleotides of the unmodified oligonucleotide. In some embodiments, the nucleic acid does not contain any insertions, deletions, inversions and/or substitutions. However, in some cases, as discussed herein, nucleic acids containing one or more insertions, deletions, inversions and/or substitutions may be suitable. In some embodiments, the nucleic acid may encode additional amino acid sequences that do not affect the function of the CAR and may or may not be translated following expression of the nucleic acid by the host cell.

Embodiments of the invention also provide isolated or purified nucleic acids comprising a nucleotide sequence that is complementary to or under stringent conditions the nucleotide sequence of any nucleic acid described herein. Hybridizing nucleotide sequences.

Nucleotide sequences that hybridize under stringent conditions can hybridize under high stringency conditions. "High stringency conditions" means that a nucleotide sequence specifically hybridizes to a target sequence (the nucleotide sequence of any nucleic acid described herein) in an amount that is detectable greater than non-specific hybridization. High stringency conditions include conditions that distinguish a polynucleotide from an exact complementary sequence, or a complementary sequence containing only a few scattered mismatches from a random sequence that happens to have some small region of matching nucleotide sequence (e.g., 3 -10 bases). Small regions of such complementarity melt more readily than full-length complementarity of 14-17 or more bases, and high stringency hybridization makes them easily distinguishable. Relatively high stringency conditions would include, for example, low salt and/or high temperature conditions, such as provided by about 0.02-0.1 M NaCl or equivalent at a temperature of about 50-70°C. Such high stringency conditions allow for few, if any, mismatches between the nucleotide sequence and the template or target strand, and are particularly suitable for detecting expression of any CAR of the invention. It is generally understood that conditions can be made more stringent by adding increasing amounts of formamide.

The invention also provides a nucleic acid composition comprising at least about 70% or greater, such as about 80%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96 A nucleic acid with a nucleotide sequence that is %, about 97%, about 98%, or about 99% identical.

In embodiments, the nucleic acids of the invention can be incorporated into recombinant expression vectors. In this regard, embodiments of the invention provide recombinant expression vectors comprising any nucleic acid of the invention. For the purposes herein, the term "recombinant expression vector" means a genetically modified oligonucleotide or polynucleotide construct when the construct contains a nucleotide sequence encoding an mRNA, protein, polypeptide, or peptide and is present in a cell in a manner sufficient to The genetically modified oligonucleotide or polynucleotide construct allows the host cell to express the mRNA, protein, polypeptide or peptide when the vector is contacted with the cell under conditions for expressing the mRNA, protein, polypeptide or peptide. The vectors of the present invention are not entirely naturally occurring. However, a portion of the carrier may be naturally occurring. The recombinant expression vector of the present invention may contain any type of nucleotides, including but not limited to DNA and RNA, which may be single-stranded or double-stranded, partially synthesized or obtained from natural sources, and may contain natural, Unnatural or altered nucleotides. Recombinant expression vectors may contain naturally occurring or non-naturally occurring internucleotide linkages, or both types of linkages. Preferably, non-naturally occurring or altered nucleotides or inter-nucleotide linkages do not hinder transcription or replication of the vector.

In embodiments, the recombinant expression vector of the invention can be any suitable recombinant expression vector, and can be used to transform or transfect any suitable host cell. Suitable vectors for the present invention include those designed for propagation and amplification or for expression or both, such as plasmids and viruses. The vector can be selected from the pUC series (Fermentas Life Sciences, Glen Burnie, MD), pBluescript series (Stratagene, La Jolla, CA), pET series (Novagen, Madison, WI), pGEX series (Pharmacia Biotech, Uppsala, Sweden) and pEX series ( Clontech, Palo Alto, CA). Phage vectors such as λGT10, λGTl1, λZapII (Stratagene), λEMBL4 and λNM1149 can also be used. Examples of plant expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and pBIN19 (Clontech). Examples of animal expression vectors include pEUK-Cl, pMAM and pMAMneo (Clontech). The recombinant expression vector can be a viral vector, such as a retroviral vector or a lentiviral vector.

The recombinant expression vector may comprise a natural or non-natural promoter operably linked to a nucleotide sequence encoding a CAR (including functional portions and functional variants thereof) or to a nucleotide sequence complementary to or hybridizing to a CAR-encoding nucleotide sequence. Nucleotide sequence. Selection of promoters, such as strong, weak, inducible, tissue-specific and development-specific, is within the ability of those skilled in the art. Similarly, combinations of nucleotide sequences and promoters are within the ordinary skill of those skilled in the art. The promoter can be a non-viral promoter or a viral promoter, such as EF1α promoter, cytomegalovirus (CMV) promoter, SV40 promoter, RSV promoter. The EF1α promoter is derived from the human targeted elongation factor 1α (EF1A) gene. In yet another aspect of the present invention, an EF1α promoter is used in the recombinant expression vector of the present invention, which has, for example, the nucleotide sequence of SEQ ID NO: 15.

In one aspect of the invention, the aforementioned chimeric antigen receptor (CAR) expression vector of the invention is provided, comprising a nucleic acid encoding the CAR, the CAR comprising: (a) an antigen-binding domain, which includes NKG2D or its Active fragment; (b) transmembrane domain and (c) intracellular signaling domain.

In one aspect of the present invention, there is also provided an expression vector of the aforementioned chimeric antigen receptor (CAR) and auxiliary protein of the present invention, which includes a nucleic acid encoding the CAR and a nucleic acid encoding an auxiliary protein, and the CAR includes: ( a) an antigen-binding domain, which includes NKG2D or an active fragment thereof; (b) a transmembrane domain and (c) an intracellular signaling domain, and the accessory protein is DAP10 or an active fragment thereof. In yet another aspect of the present invention, the expression vector of the chimeric antigen receptor (CAR) and the auxiliary protein of the present invention can have nucleic acids encoding the chimeric antigen receptor (CAR) or the auxiliary protein on different vectors. . Preferably, the expression vector of the chimeric antigen receptor (CAR) and the accessory protein of the present invention has the nucleic acid encoding the chimeric antigen receptor (CAR) and the accessory protein on the same vector.

In one aspect of the invention, the nucleotide sequence encoding the antigen-binding domain of the CAR in the expression vector of the invention includes an active fragment encoding NKG2D, for example, NKG2D.

The nucleotide sequence of fragment a.a.82-216 is, for example, the nucleotide sequence shown in SEQ ID NO: 1.

In one aspect of the invention, the nucleotide sequence encoding the transmembrane domain in the expression vector of the invention includes a nucleoside encoding the transmembrane domain of CD8 and/or CD28, preferably the transmembrane domain of CD28. The acid sequence is, for example, the nucleotide sequence of SEQ ID NO: 7.

In one aspect of the invention, the nucleotide sequence encoding the intracellular signaling domain in the expression vector of the invention includes one or more of the intracellular signaling domains encoding CD28, 4-1BB and CD3ζ. The nucleotide sequence preferably includes a nucleotide sequence encoding the intracellular signaling domain of CD28, 4-1BB and CD3ζ, and more preferably includes a nucleotide sequence encoding CD28, 4-1BB and CD3ζ in order from the amino terminus to the carboxyl terminus. Nucleic acid sequence of a protein.

Wherein, the nucleic acid encoding the intracellular signaling domain of CD28 may have, for example, the nucleotide sequence of SEQ ID NO: 9.

Wherein, the intracellular signaling domain encoding 4-1BB may have, for example, the nucleotide sequence of SEQ ID NO: 11;

Wherein, the intracellular signaling domain encoding CD3ζ may have, for example, the nucleotide sequence of SEQ ID NO: 13;

In yet another aspect of the invention, the nucleotide sequence encoding the intracellular signaling domain of the CAR has a nucleotide sequence such as SEQ ID NO: 20.

In one aspect of the invention, the expression vector of the invention also includes a nucleotide sequence encoding the hinge region between (a) the antigenic domain and (b) the transmembrane domain, preferably a nucleotide sequence encoding IgG1 , which has, for example, the nucleotide sequence of SEQ ID NO:5.

In one aspect of the invention, the expression vector of the invention also includes a nucleotide sequence encoding a leader sequence located upstream of the NKG2D or active fragment thereof, preferably a nucleotide sequence encoding a leader sequence of CD33, for example It is the nucleotide sequence shown in SEQ ID NO: 17.

In one aspect of the present invention, the expression vector of the present invention also includes a transcript upstream of the sequence encoding the guide peptide of the antigen domain, preferably the nucleotide sequence of the Kozak fragment, for example, SEQ ID NO: 16 Nucleotide sequence.

In one aspect of the present invention, the expression vector of the present invention has a promoter upstream of the sequence encoding the guide peptide of the antigenic domain, preferably an EF1α promoter, for example, the sequence of which is the nucleotide sequence of SEQ ID NO: 15 .

In one aspect of the invention, the nucleic acid encoding the DAP10 in the expression vector of the invention has the nucleotide sequence of SEQ ID NO: 3.

In one aspect of the present invention, in the expression vector of the present invention, there is also a nucleotide sequence of IRES between the fragments encoding CAR and DAP10, for example, the nucleotide sequence of SEQ ID NO: 19.

In one aspect of the invention, host cells expressing the chimeric antigen receptor (CAR) described above are also provided. In one aspect of the invention, a host cell expressing a combination of a chimeric antigen receptor (CAR) and an accessory protein as described above is also provided. In one aspect of the invention, a host cell comprising any of the previously described recombinant expression vectors is also provided.

As used herein, the term "host cell" refers to any type of cell that may contain the recombinant expression vector of the invention. The host cell can be a eukaryotic cell, such as a plant, animal, fungus, or algae, or it can be a prokaryotic cell, such as a bacterium or protozoa. Host cells can be cultured cells or primary cells, ie, isolated directly from an organism, such as a human. Host cells can be adherent cells or suspension cells, ie cells that grow in suspension. Suitable host cells are known in the art and include, for example, DH5α E. coli cells, Chinese hamster ovary cells, monkey VERO cells, COS cells, HEK293 cells, and the like. For the purpose of amplifying or replicating the recombinant expression vector, the host cell can be a prokaryotic cell, such as a DH5α cell. For the purpose of producing recombinant CAR, the host cell can be a mammalian cell. The host cell can be a human cell. The host cell can be any cell type, derived from any type of tissue and at any stage of development. For example, the host cells can be peripheral blood lymphocytes (PBL) or peripheral blood mononuclear cells (PBMC).

In one aspect of the invention, the host cell is a T cell. For the purposes herein, a T cell may be any T cell, such as a cultured T cell, such as a primary T cell or a T cell from a cultured T cell line, such as Jurkat, SupTl, etc., or a T cell obtained from a mammal. If obtained from a mammal, T cells can be obtained from a number of sources, including, but not limited to, blood, bone marrow, lymph nodes, thymus, or other tissue or fluid. T cells can also be enriched or purified. The T cells can be human T cells. The T cells may be T cells isolated from humans. T cells can be any type of T cell and can be at any developmental stage, including but not limited to CD4+/CD8+ double-positive T cells, CD4+ helper T cells such as Th 1 and Th 2 cells, CD8+ T cells such as cytotoxic T cells ), tumor infiltrating cells, memory T cells, initial T cells, etc. T cells can be CD8+ T cells or CD4+ T cells.

In one aspect of the invention, the host cell is a natural killer (NK) cell. The term "NK cells" (also known as natural killer cells) refers to a class of lymphocytes that originate in the bone marrow and play an important role in the innate immune system. NK cells provide a rapid immune response against virally infected cells, tumor cells, or other stressed cells, even in the absence of antibodies and major histocompatibility complex on the cell surface. NK cells can be isolated or obtained from commercially available sources.

The term "isolated cells" generally refers to cells that are substantially separated from other cells of the tissue. "Immune cells" include, for example, white blood cells (leukocytes) derived from hematopoietic stem cells (HSCs) produced in the bone marrow, lymphocytes (T cells, B cells, natural killer (NK) cells) and bone marrow-derived cells (neutrophils). cells, eosinophils, basophils, monocytes, macrophages, dendritic cells). "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Tregs), and γδ T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses.

The CAR substances of the present invention can be formulated into pharmaceutical compositions. In this regard, embodiments of the present invention provide a CAR, functional portion, functional variant, nucleic acid, expression vector, host cell (including a population thereof), and an antibody (including an antigen-binding portion thereof) and a pharmaceutically acceptable vector. Pharmaceutical compositions. The pharmaceutical composition of the invention containing any CAR substance of the invention may contain more than one CAR substance of the invention, such as CAR and nucleic acid, or two or more different CARs. Alternatively, the pharmaceutical composition may comprise a combination with other pharmaceutically active agents or drugs such as chemotherapeutic agents, such as asparaginase, busulfan, carboplatin, cisplatin, daunorubicin, doxorubicin, fluorouracil, gemcitabine ( The CAR substance of the present invention is a combination of gemcitabine, hydroxyurea, methotrexate, paclitaxel, rituximab, vinblastine, vincristine, and the like. In a preferred embodiment, the pharmaceutical composition comprises a host cell of the invention or a population thereof.

With respect to pharmaceutical compositions, a pharmaceutically acceptable carrier may be any of those conventionally used and is limited only by chemical-physical considerations such as solubility and lack of reactivity with the active agent and route of administration. Pharmaceutically acceptable carriers, such as vehicles, adjuvants, excipients and diluents described herein, are well known to those skilled in the art and are readily available to the public. Preferred are pharmaceutically acceptable carriers that are chemically inert toward the active agent and do not cause deleterious side effects or toxicity under the conditions of use.

Methods for preparing administrable (e.g., parenterally administrable) compositions are known or apparent to those skilled in the art and are described in more detail, for example, Remington: The Science and Practice of Pharmacy, Lippincott Williams &Wilkins; 21st Edition (May 1, 2005).

The following formulations for oral, aerosol, parenteral (eg, subcutaneous, intravenous, intraarterial, intramuscular, intradermal, intraperitoneal, and intradural) and topical administration are illustrative only and not limiting. More than one route may be used to administer the CAR agents of the invention, and in some cases a particular route may provide a more direct and effective response than another route.

For the purposes of the methods of the present invention, where a host cell or population of cells is administered, the cells may be allogeneic to the mammal or autologous to the mammal. Preferably, the cells are autologous to the mammalian species.

The mammal referred to herein may be any mammal. As used herein, the term "mammal" refers to any mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits. Mammals can be from the order Carnivora, including Felidae (cats) and Canidae (dogs). Mammals may be from the order Artiodactyla, which includes Bovids (cows) and Suids (pig), or the order Perissodactyla, which includes Equidae (horses). Mammals may be of the order Primates, Apes, or Monkeys (monkeys) or of the suborder Simimidae (humans and apes). Preferably, the mammal is human.

The pharmaceutical composition of the present invention can be used to treat or prevent cancer.

The present invention also provides the use of the previously described chimeric antigen receptor (CAR) of the present invention, or the combination of the chimeric antigen receptor (CAR) and an auxiliary protein, or the nucleic acid or the expression vector, or methods of treating or preventing cancer using said host cells. The present invention also provides the aforementioned chimeric antigen receptor (CAR) of the present invention, or a combination of the chimeric antigen receptor (CAR) and an auxiliary protein, or the nucleic acid or the expression vector, or Use of the host cell in the preparation of a medicament for treating or preventing cancer. The cancer can be any cancer, including leukemia, lymphoma, multiple myeloma or solid tumors, and in one aspect of the invention, the cancer is an NKG2D-related cancer. In the cancer, the NKG2D receptor-NKG2D ligand system is expressed in cancer cells and exerts physiological and biochemical effects. On cells of these cancers, NKG2D ligands are usually expressed, including UL16-binding protein (ULBP) or MHC class I-chain-related protein A/B (MICA/B). .

In one aspect of the invention, the cancer is liver cancer.

Description of the drawings

Figure 1. Flow cytometry analysis results of detecting the expression of NKG2D ligand on Raji cell line.

Figure 2 Expression vector construction diagram. A: pCCL-DRCAR-IRES-DAP10 expression vector; B: pHAGE-DRCAR expression vector.

Figure 3 is a schematic diagram of the nucleotide sequence and description of the insert fragment of expression vector pCCL-DRCAR-IRES-DAP10.

Figure 4 is a schematic diagram of the nucleotide sequence and description of the insert fragment of expression vector pHAGE-DRCAR.

Figure 5. Flow cytometry analysis results of plasmid co-transfected 293T cells expressing DRCAR. The far left is the control (only the lentiviral packaging plasmid is added, excluding the recombinant plasmid). In the middle, the pHAGE-DRCAR recombinant plasmid is added and the lentiviral infection is packaged. The right side shows the packaging of lentivirus infection after adding pCCL-DRCAR-IRES-DAP10 recombinant plasmid.

Figure 6 Titration of lentivirus expressing DRCAR. (A) Flow cytometry analysis results of the ratio of lentivirus expressing DRCAR in different amounts. (B) is the corresponding histogram. (C) Calculation of lentivirus titer formula and results based on flow cytometry analysis data.

Figure 7. Picture of the effect of DRCAR-T cells on killing cancer cells. Figures 7(a) to 7(c) show the effect on three liver cancer cell lines expressing NKG2D ligand. Figure 7(d) shows the effect on Raji cells not expressing NKG2D ligand. Analysis of cancer cell death rate using flow cytometry.

Figure 8. DRCAR T cells inhibit tumors in human liver cancer transplant animal models. Figure 8(a) is the experimental design diagram. Figure 8(b) is a graph showing the survival results of liver cancer transplanted mice in the experimental group injected with 4×10 ⁶ DRCAR T cells. Figure 8(c) is a graph showing the survival results of liver cancer transplanted mice in the experimental group injected with 5×10 ⁶ DRCAR T cells.

Detailed ways

Example 1 Experiments and Methods

cell

Buffy coat was obtained from the Hong Kong Red Cross Blood Transfusion Service. Peripheral blood mononuclear cells (PBMC) were isolated from the leukocyte layer by using Ficoll-Paque PLUS (GE Healthcare). T cells were isolated from PBMC by using CD3/CD28 Dynabeads (Thermo). T cells isolated from PBMC were cultured in AIM-V medium (Thermo) supplemented with 5% human serum (Sigma), 2mM L-glutamine (Thermo) and 50U/ml IL-2 (Peprotech). Original medium or expansion medium consisting of AIM-V medium supplemented with 5% human serum, 2mM L-glutamine and 300U/ml IL-2.

All the following cell lines are from ATCC, ECACC or the Chinese Academy of Sciences Cell Bank.

Human embryonic kidney epithelial cell line-293T (ATCC #CRL-3216) was cultured in DMEM medium (Thermo) supplemented with 10% FBS (Thermo), 100 U/ml penicillin and 100 U/ml streptomycin (Thermo).

Chronic myeloid leukemia cell line-K562 (ATCC #CCL-243) was cultured in IMDM medium supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin.

Liver cancer cell lines - QGY7703, SMMC7721 and BEL7402 - were cultured in RPMI1640 medium (Thermo) supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. Retroviral plasmid construction

Lentivirus packaging, concentration and purification

The third-generation lentiviral plasmids pMDLg/pRRE, pMD2.G, pRSV-Rev and the constructed expression vector were co-transfected into 293T cells at a ratio of 2:1:1:4 to produce lentivirus using the calcium phosphate transfection method. Centrifuge freshly collected or thawed lentivirus-containing supernatant at 300g for 3 minutes to exclude cell debris in the supernatant. The supernatant was filtered through a 0.45-μm micro syringe filter connected to a 30-ml syringe (TERUMO). The supernatant was centrifuged at 20,000g and 4°C for 90 minutes. After ultracentrifugation, the supernatant was removed. Add 1/10 of the starting lentivirus volume of AIM-V medium to the centrifuge tube to resuspend the pellet. Mix the lentiviral suspension by pipetting. Aliquot and store the concentrated lentivirus in a -80°C refrigerator.

Lentivirus titer determination

Seed 1 × 10 Jurkat cells into each well of a 12-well plate in 1 ml RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin and ¹⁰⁰ U/ml streptomycin. After overnight culture, different amounts (5 μl to 100 μl) of concentrated lentivirus were added to the wells. Samples were repeated three times to increase accuracy. Polybrene (Sigma) was added to a final concentration of 6ug/ul in each well. After 24 hours, cells were collected by centrifugation and resuspended in 1 ml of fresh RPMI1640 medium supplemented with 10% FBS, 100 U/ml penicillin and 100 U/ml streptomycin. After an additional 48 hours, cells were collected and the percentage of CAR-expressing Jurkat cells was determined by flow cytometry. The lentivirus titer is calculated according to the following formula.

T cell isolation, transduction and culture

A 3:1 magnetic bead to cell ratio was used to isolate CD3 ⁺ cells from 1 × ¹⁰ PBMC by using Dynabeads coated with CD3 and CD28 antibodies. Incubate the cell and magnetic bead mixture on a shaker at room temperature for 1 hour. Perform CD3 ⁺ cell enrichment using a magnet and resuspend in starting medium at ^1x10 cells/ml. After 24 hours, cells were collected by centrifugation (300xg, 3 minutes). Discard the supernatant. 5 × 10 ⁸ TU lentivirus in 500 μl AIM-V medium was added to the cells and centrifuged at 2000 × g for 2 hours. Resuspend cells in lentiviral culture and add 1.5 ml of starting medium. Place the cells back into the 6-well plate and place in an incubator (37 degrees, 5% _CO2 ). After 24 hours, transduction was performed again. After an additional 24 hours, cells were collected by centrifugation (300xg, 3 minutes) and resuspended in 2 ml of expansion medium. Place the cells back into the 6-well plate and place in an incubator (37 degrees, 5% _CO2 ). After 72 hours, cells were transferred to 100-cm culture dishes and resuspended in expansion medium at a concentration of 4 × ¹⁰ cells/ml. Transduction rates can be determined by using flow cytometry, and when T cells are sufficient, cytotoxicity assays can be performed.

Protein expression and flow cytometric analysis

To detect CAR expression on the cell surface (T cells and Jurkat cells), 1 × ¹⁰ cells were resuspended in 1 ml PBS buffer and stained with biotin goat anti-human IgG (H+L) (Jackson Lab), followed by Streptavidin-Apc (eBioscience) was used.

Cytotoxicity analysis

Target cells were collected by centrifugation and resuspended in PBS at a concentration of 1× ¹⁰ cells/ml. Stain 5ml of cells with 2.5ul Oregon Green 488 (Thermo) at 37 degrees for 20 minutes. Add 20 ml of medium to absorb excess dye. Target cells were resuspended in culture medium at a concentration of 4× ¹⁰ cells/ml.

Collect T cells by centrifugation and resuspend in the medium required for target cells at a concentration of 1.6 × ¹⁰ cells/ml.

Mix T cells and target cells at ratios of 5, 10, 20 and 40.

[Note: E: T ratio is the ratio of effector cells (T cells) to target cells. ]

The cells were incubated accordingly in the incubator for 5 to 8 hours. Cells were collected by centrifugation and resuspended in 500 μl of 7-AAD solution (1 μg/ml). Cells were incubated on ice for 30 min. Mortality was analyzed by flow cytometry (7-AAD: excitation wavelength 561 nm, emission wavelength 670 nm).

Example 2 Expression of NKG2D ligands on various cancer cell lines

The expression of NKG2D ligands (human MICA/B and human ULBP1-ULBP6) was detected on different cancer cell lines to determine whether CARs with NKG2D as the antigenic domain can be used to kill these cell lines.

To detect human MICA/B, 1× ¹⁰ cells to be tested were resuspended in 0.5 ml PBS buffer and treated with monoclonal mouse anti-human MICA/B (R&D Cat#MAB13001), followed by biotinylated goat anti-human MICA/B. Mouse IgG (H+L) and streptavidin-APC staining.

To detect human ULBP2/5/6, 1 × 10 ⁶ cells to be tested were resuspended in 0.5 ml PBS buffer and treated with monoclonal mouse anti-human ULBP2/5/6 (R&D Cat#MAB1298), followed by biological Primed goat anti-mouse IgG (H+L) and streptavidin-APC staining.

To detect human ULBP1, 1 × ¹⁰ cells to be tested were resuspended in 0.5 ml PBS buffer and treated with monoclonal mouse anti-human ULBP1 (R&D Cat#MAB1380), followed by biotinylated goat anti-mouse IgG (H +L) and streptavidin-APC staining.

To detect human ULBP3, 1 × ¹⁰ cells to be tested were resuspended in 0.5 ml PBS buffer and treated with monoclonal mouse anti-human ULBP3 (R&D Cat#MAB1517), followed by biotinylated goat anti-mouse IgG (H +L) and streptavidin-APC staining.

To detect human ULBP4, 1 × ¹⁰ cells to be tested were resuspended in 0.5 ml PBS buffer and treated with monoclonal mouse anti-human ULBP4 (R&D Cat#AF6285), followed by biotinylated bovine anti-goat IgG (H+ L) and streptavidin-APC staining.

The following liver cancer cell lines were tested to detect the expression of NKG2D ligands (human MICA/B and human ULBP1-ULBP6):

Liver cancer cell lines-QGY7703, SMMC7721 and BEL7402

Experimental results proved that the NKG2D ligand was expressed in the above three cancer cell lines. The above three cancer cell lines were tested as target cells in the following CAR T cell killing experiment.

In addition, as shown in Figure 1, Raji cells do not express NKG2D ligands (human MICA/B and human ULBP1-ULBP6). Raji cells were used as negative controls in CAR T cell killing experiments.

Example 3 Construction of lentiviral vector expressing DRCAR

1.Construct pCCL-DRCAR-IRES-DAP10

pCCL-DRCAR-IRES-DAP10 has the structure shown in Figure 2A. pCCL-DRCAR-IRES-DAP10 was constructed by the following method.

The nucleotide sequences of the nucleic acid inserts encoding DRCAR and DAP10 were synthesized as shown in Figure 3 . The inserted fragment includes from 5' end to 3' end: 1. HpaI restriction site; 2. EF1α promoter; 3. Kozak; 4. CD33 leader sequence; 5. a.a.82-216 fragment of NKG2D; 6. As IgG1H in the hinge region; 7. CD28 transmembrane domain; 8. Intracellular signaling domain of CD28; 9.4-1BB intracellular signaling domain; 10. Intracellular signaling domain of CD3ζ; 11. Connecting fragment; 12. IRES; 13. Connection fragment; 14. DAP10; 15. Sal I restriction site.

The above insert fragment and plasmid Pax5 (Addgene, plasmid #35003) were double digested with restriction endonucleases HpaI and Sal I. After the digested products were recovered from the gel, they were ligated with T4 ligase at 16°C overnight. After ligation, the cells were transformed into competent E. coli cells and spread on plates. The next day, single clones were picked for double enzyme digestion and sequencing identification to obtain pCCL-DRCAR-IRES-DAP10.

2.Construct pHAGE-DRCAR

pHAGE-DRCAR has the structure shown in Figure 2B. pHAGE-DRCAR was constructed by the following method.

The nucleic acid insert encoding DRCAR was synthesized with the nucleotide sequence shown in Figure 4 . The inserted fragment includes from 5' end to 3' end: 1. HpaI restriction site; 2. EF1α promoter; 3. Kozak; 4. CD33 leader sequence; 5. a.a.82-216 fragment of NKG2D; 6. As IgG1H in the hinge region; 7. CD28 transmembrane domain; 8. Intracellular signaling domain of CD28; 9.4-1BB intracellular signaling domain; 10. Intracellular signaling domain of CD3ζ; 11. Sal I enzyme cut point.

The above insert fragment and plasmid Pax5 were double digested with restriction endonucleases HpaI and Sal I. After the digested products were recovered by gel cutting, they were ligated with T4 ligase at 16°C overnight. After ligation, the E. coli cells were transformed into competent cells and spread on plates. The next day, single clones were picked for double enzyme digestion and sequencing identification to obtain pHAGE-DRCAR.

3. Generation of lentiviral vectors

pHAGE-DRCAR and pCCL-DRCAR-IRES-DAP10 were used as expression plasmids respectively, and the third-generation lentiviral plasmids pMDLg/pRRE, pMD2.G, and pRSV-Rev were co-transfected into 293T cells to prepare corresponding lentiviral vectors.

The ability of lentivirus to express DRCAR was calculated according to the method described in Example 1. Figure 5 is a flow cytometry analysis result of plasmid co-transfected 293T cells expressing DRCAR. The results showed that compared with pHAGE-DRCAR, the lentivirus containing pCCL-DRCAR-IRES-DAP10 had a significantly higher DRCAR expression level.

The titer of the lentivirus containing pCCL-DRCAR-IRES-DAP10 was calculated according to the method described in Example 1. Figure 6 shows that the titer of pCCL-DRCAR-IRES-DAP10 packaging lentivirus is approximately 2×10 ⁷ TU/ml. Compared with pHAGE-DRCAR, the lentivirus containing pCCL-DRCAR-IRES-DAP10 has a higher lentivirus titer.

Example 4 DRCAR-T cells kill cancer cells in vitro

According to the method described in Example 1, T cells were extracted and obtained from human PBMC. Then, the lentivirus containing pCCL-DRCAR-IRES-DAP10 prepared in Example 3 was used to transfect T cells.

Cytotoxicity assay was performed according to the method described in Example 1 to examine the specific cytotoxic effect of DRCAR-expressing T cells on various tumor cells. Among them, T cells containing pCCL-DRCAR-IRES-DAP10 and T cells not transfected with lentivirus were used as effector cells, and 21 types of cancer cells were used as target cells.

The Raji cell line, which does not express NKG2D ligand, was used as a negative control.

The results are shown in Figure 7(a)-Figure 7(c). Under the same experimental conditions, compared with normal T cells without DRCAR, T cells expressing DRCAR were able to induce the death of significantly more target tumor cells. The tumor cells include: liver cancer cell lines-QGY7703, SMMC7721 and BEL7402.

As shown in Figure 7(d), in the negative control of the Raji cell line that does not express NKG2D ligand, DRCAR-T cells did not show any difference from the control natural T cells.

Example 5 DRCAR T cells inhibit tumors in human liver cancer transplant animal model

Figure 8(a) is the experimental design diagram.

A total of 20 experimental mice (NSG mice, 6-8 weeks old, provided by the Animal Room of Hong Kong University of Science and Technology) were divided into DRCAR T cell treatment group (12 randomly selected) and control group (8). All mice were injected with liver cancer cells SMMC7721 (1 × 10 ⁶ cells) on day 0 of the experiment, and each group was injected with the same number of DRCAR T cells (treatment group) or three times at weeks 2, 5, and 7 of the experiment. T cells (control group). The tumor growth and survival of the mice were observed every day, and the data were recorded. Mice at the end of the experiment were characterized by death, or weight loss of 20% or more, sunken eyes or closed eyelids, unresponsiveness or detachment.

experiment one:

Inject 4 × 10 ⁶ DRCAR T cells (treatment group) or T cells (blank group) three times in the 2nd, 5th and 7th weeks of the experiment.

The experimental results are shown in Figure 8(b).

Experiment 2:

Inject 5×10 ⁶ DRCAR T cells (treatment group) or T cells (blank group) three times in the 2nd, 5th and 7th weeks of the experiment.

The experimental results are shown in Figure 8(c).

It can be concluded from the experimental results that compared with the control group, the DRCAR T cells of the present invention can protect the survival of cancer-stricken animals more effectively than natural T cells that do not contain DRCAR.

The above experimental results show that the DRCAR and DRCAR-T cells with NKG2D antigen receptor structure provided by the present invention can effectively recognize cancer cells with NKG2D ligands and activate tumor cell-specific anti-tumor cell immune responses and killing. related tumor cells. Experiments also prove that the DRCAR and DRCAR-T cells provided by the present invention can kill various liver cancer cells in a broad spectrum, and their liver cancer inhibitory effect has been proven in animals.

The above is a description of the present invention, and it cannot be regarded as a limitation of the present invention. Unless otherwise indicated, the practice of the present invention will employ conventional techniques of organic chemistry, polymer chemistry, biotechnology, etc. It will be apparent that the present invention may be carried out in other ways than those specifically described in the above illustration and examples. Other aspects and modifications within the scope of the invention will be apparent to those skilled in the art to which this invention belongs. Many modifications and variations are possible in light of the teachings of this invention and are therefore within the scope of this invention. the following

Unless otherwise stated, the unit "degree" for temperature appearing in this article refers to degrees Celsius, that is, °C.

The following documents are incorporated by reference in this application in full.

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cgggtgccta gagaaggtgg cgcggggtaa actgggaaag tgatgtcgtg tactggctcc 120

gcctttttcc cgagggtggg ggagaaccgt atataagtgc agtagtcgcc gtgaacgttc 180

tttttcgcaa cgggtttgcc gccagaacac ag 212

<210> 16

<211> 18

<212> DNA

<213> Artificial sequence()

<400> 16

gaaggagaggccaccatg 18

<210> 17

<211> 51

<212> DNA

<213> person()

<400> 17

atgccgctgc tgctactgct gcccctgctg tgggcagggg ccctggctat g 51

<210> 18

<211> 17

<212> PRT

<213> person()

<400> 18

Met Pro Leu Leu Leu Leu Leu Pro Leu Leu Trp Ala Gly Ala Leu Ala

1 5 10 15

Met

<210> 19

<211> 578

<212> DNA

<213> Artificial sequence()

<400> 19

ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt gtgcgtttgt 60

ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc ggaaacctgg 120

ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag gaatgcaagg 180

tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac aaacaacgtc 240

tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc tctgcggcca 300

aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc acgttgtgag 360

ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca aggggctgaa 420

ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt gcacatgctt 480

tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg gggacgtggt 540

tttcctttga aaaacacgat gataatatgg ccacacat 578

<210> 20

<211> 591

<212> DNA

<213> Artificial sequence()

<400> 20

gtgaggagta agaggagcag gctcctgcac agtgactaca tgaacatgac tccccgccgc 60

cccgggccca cccgcaagca ttaccagccc tatgccccac cacgcgactt cgcagcctat 120

cgctccaaac ggggcagaaa gaaactcctg tatatattca aacaaccatt tatgagacca 180

gtacaaacta ctcaagagga agatggctgt agctgccgat ttccagaaga agaagaagga 240

ggatgtgaac tgagagtgaa gttcagcagg agcgcagagc cccccgcgta ccagcagggc 300

cagaaccagc tctataacga gctcaatcta ggacgaagag aggagtacga tgttttggac 360

aagagacgtg gccgggaccc tgagatgggg ggaaagccga gaaggaagaa ccctcaggaa 420

ggcctgtaca atgaactgca gaaagataag atggcggagg cctacagtga gattggggatg 480

aaaggcgagc gccggagggg caaggggcac gatggccttt accagggtct cagtacagcc 540

accaaggaca cctacgacgc ccttcacatg caggccctgc cccctcgcta a 591

Claims (4)

1. The use of a host cell for preparing a medicament for treating or preventing liver cancer, the host cell being a T cell derived from a T cell isolated from a subject, comprising a nucleic acid or an expression vector comprising the nucleic acid, Wherein the nucleic acid includes a nucleic acid encoding a chimeric antigen receptor (CAR) and a nucleic acid encoding DAP10, the CAR comprising: (a) an antigen-binding domain, which includes a.a.82-216 fragment of NKG2D, which is an amino acid sequence such as A polypeptide of amino acids 82-216 as shown in SEQ ID NO: 2; (b) a transmembrane domain, the amino acid sequence of which is as shown in SEQ ID NO: 8; and (c) in order from the amino terminus to the carboxyl terminus: The intracellular signaling domains of CD28, 4-1BB and CD3ζ, where the amino acid sequence of the CD28 intracellular domain is shown in SEQ ID NO: 10, and the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 12 As shown, the amino acid sequence of the CD3ζ intracellular domain is shown in SEQ ID NO: 14; the amino acid sequence of the DAP10 is shown in SEQ ID NO: 4; and, the nucleic acid fragment encoding the CAR and the nucleic acid fragment encoding the DAP10 fragments with IRES in between; The nucleic acid further includes a nucleic acid encoding a hinge region between (a) the antigenic domain and (b) the transmembrane domain, and the hinge region is IgG1H, the amino acid sequence of which is shown in SEQ ID NO: 6, The nucleic acid further includes a nucleic acid encoding a leader fragment located upstream of the NKG2D fragment. The leader fragment is the leader fragment of CD33, and its amino acid sequence is shown in SEQ ID NO: 18. 2. The use according to claim 1, wherein the nucleotide sequence encoding the (a) antigen-binding domain of the CAR is shown in SEQ ID NO: 1; the (b) transmembrane domain encoding the CAR The nucleotide sequence is shown in SEQ ID NO: 7; the nucleotide sequence encoding the intracellular signaling domain of (c) CD28, 4-1BB and CD3ζ from the amino terminus to the carboxyl terminus of the CAR As shown in SEQ ID NO: 9, SEQ ID NO: 11 and SEQ ID NO: 13 respectively; the nucleotide sequence encoding the DAP10 is shown in SEQ ID NO: 3; and, the nucleic acid fragment encoding the CAR and The nucleotide sequence of the IRES between the nucleic acid fragments encoding the DAP10 is shown in SEQ ID NO: 19; The nucleotide sequence encoding the hinge region between (a) the antigenic domain and (b) the transmembrane domain is shown in SEQ ID NO: 5, and the nucleotide sequence encoding the leader sequence of CD33 is shown in SEQ ID NO:17 shown. 3. The use according to claim 2, wherein the nucleotide sequence encoding the intracellular signaling domain of (c) CD28, 4-1BB and CD3ζ from the amino terminus to the carboxyl terminus of the CAR is such as SEQ. ID NO:20 is shown. 4. The use according to claim 1, wherein the expression vector is a lentiviral vector.